Have you checked the instructions that came with the kit?

The recommended annealing temperature for the U6 primer in microRNA qPCR kits with Poly A tailing is generally around 60°C. Specifically, for similar Universal reverse primers used in Poly A tailing methods and qPCR detection, an annealing temperature of approximately 60°C is common practice.

The answer to this question comes from MedChemExpress Technical Support. [email protected]

Usually we get amplification at 60 while using SYBR Master mix in qRT. In simple pcr it is subject to Taq polymerase

The annealing temperature is kit-dependent, so you should confirm it in the manufacturer’s protocol. For most commercial microRNA RT-qPCR kits, the U6 snRNA primer anneals in the 55–60 °C range. Standard cycling conditions are typically 95 °C for denaturation followed by 55 °C for annealing/extension. If you are using GeneCopoeia’s MicroRNA qPCR III kit, the recommended annealing temperature is 55 °C.